PRMT1 protein degradation agent modified by taking adamantane as hydrophobic group and application of PRMT1 protein degradation agent

Abstract

The invention discloses a PRMT1 protein degradation agent modified with adamantane as a hydrophobic group and application of the PRMT1 protein degradation agent, and belongs to the field of medicinal chemistry. The compound represented by the formula I can effectively degrade PRMT1 protein in tumor cells, has a wide application prospect in preparation of drugs for treating or preventing cancers, and is especially suitable for preparation of drugs for targeted treatment of breast cancer. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a PRMT1 protein degradation agent modified with adamantane as a hydrophobic group, and a preparation method and application thereof. Background Art

[0002] Histone post-translational modification is an important part of epigenetics, mainly including methylation, acetylation, phosphorylation, ubiquitination and glycosylation, etc. Among them, methylation plays an important role in various cell life activities such as cell signal transduction, DNA damage and repair, and transcription regulation.

[0003] Protein methyltransferases are key enzymes for histone methylation modification and can be divided into two categories: protein lysine methyltransferases and protein arginine methyltransferases. There are 9 types of protein arginine methyltransferases (PRMTs) (PRMT1-9), which can be divided into three types according to the different products obtained by their catalysis. Type I includes PRMT1 / 2 / 3 / 4 / 6 / 8, which catalyze asymmetric dimethylation and monomethylation of arginine; Type II includes PRMT5 / 9, which catalyze symmetric dimethylation and monomethylation of arginine; Type III only has PRMT7, which only catalyzes monomethylation of arginine. Among them, PRMT1 is the most important type I PRMT in mammalian cells. The arginine methylation involved in it affects multiple cellular processes such as cell signal transduction, DNA damage and repair, transcriptional regulation, and RNA metabolism, and plays an important role in regulating the occurrence and development of various human tumors. Many studies have shown that PRMT1 is abnormally expressed in many cancers, including breast cancer, colorectal cancer, lung cancer, and pancreatic cancer. PRMT1 is overexpressed in most tumors, which suggests that PRMT1 may become a potential biomarker or therapeutic target in tumor treatment.

[0004] All the PRMT1 inhibitors reported so far are pan-inhibitors with poor selectivity for PRMT1. The only one that entered clinical trials, GSK3368715, had its clinical trials terminated due to thrombotic toxicity and low clinical efficacy.

[0005] Targeted protein degradation (TPD) technology has promoted a paradigm shift in treatment strategies and provided an innovative approach to drug design. Compared with traditional small molecule drugs, TPD drugs have a wider range of action, can target "undruggable" targets, can better overcome drug resistance problems, and can improve selectivity for target proteins. Hydrophobic tags (HyT) are bifunctional TPD molecules, which are composed of a lipophilic small molecule tag group and a small molecule ligand of the target protein connected by a certain chemical chain. The hydrophobic tag technology increases the hydrophobicity of the target protein surface, thereby inducing instability and misfolding of the target protein, which is then degraded by the proteasome. The hydrophobic tag strategy has been widely used in the design of degraders for multiple targets, such as EZH2, estrogen receptor (ER), and androgen receptor (AR), among which the hydrophobic tag-based degrader for ER has been approved for the treatment of breast cancer. Protein degraders for PRMT1 are expected to improve the selectivity and efficacy of PRMT1, reduce clinical adverse reactions, and improve clinical treatment effects. Summary of the invention

[0006] The purpose of the present invention is to provide a hydrophobic tag bifunctional molecule having the activity of degrading PRMT1 protein and a preparation method and use thereof.

[0007] In a first aspect of the present invention, there is provided a compound of formula I or a pharmaceutically acceptable salt thereof,

[0008]

[0009] Linker is selected from:

[0010] Where Z1 is connected to the benzene ring,

[0011] Z1 and Z2 are each independently NHC(O) or C(O)NH;

[0012] n is a positive integer from 1 to 7.

[0013] In another preferred embodiment, Z1 is -NH-CO-, wherein NH is connected to the benzene ring.

[0014] In another preferred embodiment, Z2 is -NH-CO-, wherein CO is connected to the -CH2-adamantane ring.

[0015] In another preferred embodiment, n is 1, 2, 3, 4, 5 or 6.

[0016] In another preferred embodiment, Linker is n is 2, 3, 4, 5 or 6.

[0017] In another preferred embodiment, Linker is selected from:

[0018]

[0019] In another preferred embodiment, the compound is selected from the following group:

[0020]

[0021]

[0022] Preferably, the molecular structure of the PRMT1 degrader is:

[0023]

[0024] The compound can effectively induce PRMT1 protein degradation in a dose- and time-dependent manner.

[0025] The second aspect of the present invention provides a pharmaceutical composition comprising:

[0026] The compound of the first aspect or a pharmaceutically acceptable salt thereof; and

[0027] A pharmaceutically acceptable carrier.

[0028] The third aspect of the present invention provides the use of the compound or pharmaceutically acceptable salt thereof according to the first aspect or the pharmaceutical composition according to the second aspect for preparing a PRMT1 degrading agent; or

[0029] Used for preparing drugs for treating diseases related to abnormal expression of PRMT1 protein.

[0030] In another preferred embodiment, the compound or pharmaceutically acceptable salt thereof described in the first aspect or the pharmaceutical composition described in the second aspect is used to prepare a drug for treating a disease associated with overexpression of PRMT1 protein.

[0031] In another preferred embodiment, the compound or pharmaceutically acceptable salt thereof according to the first aspect or the pharmaceutical composition according to the second aspect is used to prepare a drug for treating cancer associated with abnormal expression of PRMT1 protein.

[0032] In another preferred embodiment, the compound or pharmaceutically acceptable salt thereof according to the first aspect or the pharmaceutical composition according to the second aspect is used to prepare a drug for treating cancer associated with overexpression of PRMT1 protein.

[0033] In another preferred embodiment, the disease is cancer.

[0034] In another preferred embodiment, the cancer is selected from the group consisting of breast cancer, gastric cancer, lung cancer, melanoma, and acute myeloid leukemia.

[0035] In another preferred embodiment, the cancer is breast cancer.

[0036] The third aspect of the present invention provides an intermediate for preparing the compound or a pharmaceutically acceptable salt thereof according to the first aspect, the structure of which is:

[0037]

[0038] The invention obtains a protein degradation agent modified with adamantane as a hydrophobic group. The degradation agent can be used to prepare a PRMT1 degradation agent and is particularly suitable for the development of drugs for treating breast cancer.

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results of Western blotting for detecting the PRMT1 protein degradation activity of compounds CM1-101 to CM1-112 in MCF-7 cells are shown, wherein 101-112 in the figure are abbreviations of compound numbers CM1-101 to CM1-112.

[0041] Figure 2 Western blotting was used to detect the PRMT1 protein degradation activity of compound CM1-112 in MCF-7 cells. The results showed that as the concentration of compound CM1-112 increased, the immunoblotting in the imaging became lighter and the protein degradation rate became higher.

[0042] Figure 3 Western blotting was used to detect the PRMT1 protein degradation activity of compound CM1-112 in MDA-MB-231 cells. The results showed that as the concentration of compound CM1-112 increased, the immunoblot in the imaging became lighter and the protein degradation rate became higher.

[0043] Figure 4 The results show the inhibitory effect of compound CM1-112 on the proliferation of MCF-7 tumor cells.

[0044] Figure 5 Immunoblotting experiments were used to detect the inhibitory effects of compounds on asymmetric dimethylation of H4R3, a direct substrate of PRMT1, and asymmetric dimethylation of arginine in all intracellular proteins.

[0045] Figure 6 The results of staining cell clones with crystal violet reagent are shown. Figure 7The inhibitory ability of compound CM1-112 on the proliferation of MDA-MB-231 tumor cells was tested by measuring cell confluence. The results showed that compound CM1-112 could effectively inhibit the proliferation of MDA-MB-231 cells. DETAILED DESCRIPTION

[0046] The inventors of the present application have conducted extensive and in-depth research and developed a protein degrader modified with adamantane as a hydrophobic group, which can be used to prepare a PRMT1 degrader, and is particularly suitable for the development of drugs for treating breast cancer. On this basis, the present invention was completed.

[0047] the term

[0048] In the present invention, the halogen is F, Cl, Br or I.

[0049] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0050] In the present invention, the term "C1-C6" refers to having 1, 2, 3, 4, 5 or 6 carbon atoms, "C1-C8" refers to having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, and so on. "3-8 membered" refers to having 3, 4, 5, 6, 7 or 8 ring atoms, and so on.

[0051] In the present invention, the term "alkyl" refers to a saturated linear or branched hydrocarbon moiety. For example, the term "C1-C6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.; preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0052] "Alkylene" refers to a straight or branched chain saturated aliphatic group having a specified number of carbon atoms and connecting at least two other groups, i.e., a divalent hydrocarbon group. The two groups connected to the alkylene group can be connected to the same or different atoms on the alkylene group. For example, a straight chain alkylene group can be -(CH2) n -divalent radical, wherein n is 1, 2, 3, 4, 5 or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene. Alkylene groups may be substituted or unsubstituted.

[0053] Pharmaceutically acceptable salts of the present invention can be salts formed by negatively charged groups on anions and compounds of formula I. Suitable negatively charged groups are chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronide, lactate, glutarate or maleate. Similarly, salts can be formed by negatively charged groups on cations and compounds of formula I. Suitable cations include sodium ions, potassium ions, magnesium ions, calcium ions and ammonium ions, such as tetramethylammonium ions.

[0054] In another preferred embodiment, "pharmaceutically acceptable salt" refers to the salts formed by the compound of formula I with an acid selected from the following group: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid; or the sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the compound of formula I with an inorganic base; or the methylamine salt, ethylamine salt or ethanolamine salt formed by the compound of general formula I with an organic base.

[0055] Pharmaceutical composition

[0056] The present invention also provides a pharmaceutical composition, which comprises an active ingredient within a safe and effective amount range and a pharmaceutically acceptable carrier.

[0057] The "active ingredient" described in the present invention refers to the compound of formula I described in the present invention.

[0058] The "active ingredient" and pharmaceutical composition described in the present invention can be used as a PRMT1 degrader.

[0059] The "active ingredient" and pharmaceutical composition of the present invention are used to prepare drugs for treating diseases associated with abnormal expression of PRMT1 protein. The disease associated with abnormal expression of PRMT1 protein is cancer associated with abnormal expression of PRMT1 protein.

[0060] The "active ingredient" and pharmaceutical composition of the present invention are used to prepare drugs for treating diseases associated with overexpression of PRMT1 protein. The disease associated with abnormal expression of PRMT1 protein is cancer associated with overexpression of PRMT1 protein.

[0061] The cancer is selected from: breast cancer, gastric cancer, lung cancer, melanoma, colon cancer, lymphoma (such as diffuse large B-cell lymphoma, follicular lymphoma), leukemia (such as acute myeloid leukemia), multiple myeloma, mesothelioma, malignant rhabdoid tumor, hepatocellular carcinoma, prostate cancer, bile duct and gallbladder cancer, bladder cancer; brain tumors, including neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma; cervical cancer, endometrial cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcoma, cardiovascular disease, neurodegenerative diseases, malaria, AIDS, gout, diabetes, renal failure, chronic lung disease.

[0062] "Safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably, 10-200 mg of active ingredient per dose. Preferably, the "one dose" is one tablet.

[0063] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0064] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0065] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0066] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.

[0067] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0068] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0069] The compounds of the present invention can be administered alone or in combination with other therapeutic drugs (such as anti-tumor drugs).

[0070] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0071] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions (such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions recommended by the manufacturer. The experimental materials used in the following examples, if not otherwise specified, are purchased from conventional biochemical reagent stores. The nuclear magnetic resonance spectrum is measured by a Bruker 400 nuclear magnetic resonance spectrometer, and the mass spectrum is measured by an Agilent liquid chromatography-time of flight mass spectrometer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0072] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0073] Example 1

[0074] Preparation of compound CM1-112

[0075]

[0076]

[0077] Preparation of compound Ma: Under nitrogen protection, p-nitrobenzaldehyde (3.00 g, 19.85 mmol) was dissolved in tetrahydrofuran (50 mL), and sodium hydride (0.52 g, 21.84 mmol) and diethylphosphoroacetate (4.59 g, 21.84 mmol) were added respectively in an ice bath, and the mixture was reacted at room temperature for 24 hours. After the reaction was completed by TLC monitoring, the solvent was dried and extracted three times with ethyl acetate (20 mL). The organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain a crude product to obtain the desired compound Ma (4.985 g, yield 121.3%) as a yellow solid, which was directly used in the next step without further purification.

[0078] Compound Ma was tested, and the test results are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.25(d,J=8.8Hz,2H),8.02(d,J=8.8Hz,2H),7.78(d,J=16.1Hz,1H),6.87(d,J=16.0Hz,1H),3.76(s,3H).MS(ESI):m / z[M+H] + 208.1.

[0079]

[0080] Preparation of compound Mb: Under ice bath conditions, sodium hydride (1.39 g, 57.92 mmol), compound Ma (3.99 g, 19.31 mmol) and p-toluenesulfonylmethyl isocyanide (4.55 g, 23.17 mmol) were dissolved in tetrahydrofuran / N,N-dimethylformamide DMF (100 mL, v:v=1:1), and the reaction was carried out at room temperature for 2 hours. After the reaction was completed by TLC monitoring, it was extracted three times with ethyl acetate (20 mL), the organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain a crude product to obtain the desired compound Mb (4.00 g, yield 85.2%) as a reddish-brown solid, which was directly used in the next reaction without further purification.

[0081] Compound Mb was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ8.19–8.11(m,2H),7.62–7.53(m,2H),7.48(dd,J=3.1,2.2Hz,1H),6.85(t,J=2.4Hz,1H),3.69(d,J=7.9Hz,3H).MS(ESI):m / z[M+H] + 247.1.

[0082]

[0083] Preparation of compound Mc: Under normal temperature conditions, compound Mb (4.00 g, 16.25 mmol), Boc anhydride (di-tert-butyl dicarbonate, 3.90 g, 17.87 mmol), triethylamine (2.30 g, 22.74 mmol) and 4-dimethylaminopyridine DMAP (198 mg, 0.16 mmol) were dissolved in dichloromethane DCM (80 mL) and reacted at room temperature for 5 hours. After the reaction was completed by TLC monitoring, the solvent was dried, and dichloromethane (20 mL) was extracted three times. The organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain a crude product, which was obtained by silica gel chromatography to obtain the desired compound Mc (4.43 g, yield 64.6%) as a light yellow solid.

[0084] The compound Mc was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ8.26–8.19(m,2H),7.94(d,J=2.4Hz,1H),7.66–7.59(m,2H),7.33(d,J=2.4Hz,1H),3.78(s,3H),1.64(s,9H).MS(ESI):m / z[M+H] + 347.1.

[0085]

[0086] Preparation of compound Md: Under nitrogen protection, compound Mc (4.43 g, 28.92 mmol) was dissolved in anhydrous tetrahydrofuran (60 mL), and diisobutylaluminum hydride DIBALH (8.26 g, 72.29 mmol) was slowly added dropwise within 30 minutes at -78 ° C, and reacted at -78 ° C for 6 hours. After the reaction was completed by TLC monitoring, it was quenched with methanol (5 mL), and a saturated sodium potassium tartrate solution (100 mL) was added to the reaction solution and stirred for 2 hours. It was extracted three times with ethyl acetate (20 mL), the organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain a crude product, and the desired compound Md (1.83 g, yield 45.15%) was obtained by silica gel chromatography as a yellow solid.

[0087] The compound Md was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ8.18(d,J=9.0Hz,2H),7.69(d,J=9.0Hz,2H),7.42(s,1H),7.30(s,1H),4.57(s,2H),1.56(s,9H).MS(ESI):m / z[M+H] + 319.1.

[0088]

[0089] Preparation of compound Me: Under normal temperature conditions, compound Md (1.83 g, 5.81 mmol) was dissolved in dichloromethane (60 mL), and Dess-Martin periodinane (2.71 g, 6.39 mmol) was added, and stirred at room temperature for 2 hours. After the reaction was completed by TLC monitoring, the reaction solution was filtered through a Buchner funnel covered with diatomaceous earth, and the filter cake was washed three times with dichloromethane. Anhydrous sodium sulfate was added to the filtrate for drying, and the crude product was obtained after the solvent was removed by concentration under reduced pressure. The desired compound Me (1.68 g, yield 92.39%) was obtained by silica gel chromatography as a yellow solid.

[0090] Compound Me was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ9.93 (s, 1H), 8.25 (d, J = 9.0Hz, 2H), 7.98 (d, J = 2.4Hz, 1H) ,7.72(d,J=9.0Hz,2H),7.45(d,J=2.4Hz,1H),1.66(s,9H).MS(ESI):m / z[M+H] + 317.1.

[0091]

[0092] Preparation of compound Mf: Under normal temperature conditions, compound Me (1.68 g, 5.31 mmol), sodium triacetoxyborohydride (1.69 g, 7.97 mmol) and tert-butyl 2-(methylamino)ethylcarbamate (1.11 g, 6.37 mmol) were dissolved in dichloromethane (50 mL) and reacted at room temperature for 24 hours. After the reaction was completed by TLC monitoring, the reaction solution was filtered through a Buchner funnel covered with diatomaceous earth, the filter cake was washed three times with dichloromethane, anhydrous sodium sulfate was added to the filtrate for drying, and the crude product was obtained after the solvent was removed by concentration under reduced pressure. The desired compound Mf (1.88 g, yield 74.59%) was obtained by silica gel chromatography as a light yellow solid.

[0093] Compound Mf was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ8.27–8.21(m,2H),7.83(d,J=8.8Hz,2H),7.46(d,J=2.4Hz,1H),7.21(d,J=2.5Hz,1H),3.4 0(s,2H),3.21(d,J=5.1Hz,2H),2.49(t,J=5.9Hz,2H),2.19(s,3H),1.63(s,9H),1.43(s,9H).MS(ESI):m / z[M+H] + 475.2.

[0094]

[0095] Preparation of compound Mg: Under normal temperature conditions, compound Mf (1.88 g, 3.96 mmol) was dissolved in methanol (50 mL), 10% palladium carbon (50 mg) was added, hydrogen was introduced, and the mixture was reacted at room temperature for 10 hours. After the reaction was completed by TLC monitoring, the reaction solution was filtered through a Buchner funnel covered with diatomaceous earth, the filter cake was washed three times with methanol, anhydrous sodium sulfate was added to the filtrate for drying, and the crude product was obtained after the solvent was removed by concentration under reduced pressure. The desired compound Mg (1.30 g, yield 76.86%) was obtained by silica gel chromatography as a yellow solid.

[0096] The compound Mg was tested, and the test results are as follows: 1H NMR (400MHz, DMSO-d6) δ7.26(d,J=8.4Hz,2H),7.18–7.12(m,2H),6.56(d,J=8.5Hz,3H),5.06(s,2H),3.27(s ,2H),3.03(q,J=5.9Hz,2H),2.37(t,J=7.1Hz,2H),2.14(s,3H),1.56(s,9H),1.36(s,9H).MS(ESI):m / z[M+H] + 445.2.

[0097]

[0098] Preparation of compound M-12: 1-adamantaneacetic acid (0.26 g, 1.37 mmol) was dissolved in DMF (5 mL) under ice bath conditions, N, N-diisopropylethylamine DIPEA (0.26 g, 2.05 mmol), 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate HATU (0.78 g, 2.05 mmol) and 15-amino-4,7,10,13-tetraoxapentadecanoic acid tert-butyl ester (0.5 g, 1.37 mmol) were added, and the mixture was reacted at room temperature for 1 hour. After the reaction was completed by TLC monitoring, it was extracted three times with ethyl acetate (5 mL), the organic phase was collected and washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and the concentrated product was dissolved in dichloromethane (6 mL), trifluoroacetic acid (3 mL) was added, and the reaction was carried out at room temperature for 5 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the desired compound M-12 (0.7 g, yield 94.5%) was obtained by reverse phase chromatography as a colorless transparent oil.

[0099] Compound M-12 was tested, and the test results were as follows: 1 H NMR (400MHz, DMSO-d6) δ12.18(s,1H),7.74(t,J=5.7Hz,1H),3.59(t,J=6.4Hz,2H),3.50(d,J=4.6Hz,14H) ,3.37(s,3H),3.17(q,J=5.7Hz,2H),2.44(t,J=6.3Hz,2H),1.90(s,3H),1.82(s,2H),1.69–1.49(m,12H).

[0100] Preparation of compound CM1-112: Under ice bath conditions, compound M12 (250 mg, 514.81 μmol), HATU (0.294 g, 772.21 μmol), DIPEA (0.100 g, 772.21 μmol) and compound Mg (0.228 g, 514.81 μmol) were dissolved in DMF (3 mL) and reacted at room temperature for 1 hour. After the reaction was completed by TLC monitoring, it was extracted three times with ethyl acetate (5 mL), the organic layer was collected and washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and the collected compound was dissolved in dichloromethane (4 mL), trifluoroacetic acid (2 mL) was added, and the reaction was carried out at room temperature for 30 minutes. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, and the desired compound CM1-112 (45 mg, yield 12.29%) was separated by a reverse phase chromatography column as a brown oily liquid.

[0101] Compound CM1-112 was tested, and the test results are as follows: 1 H NMR (400MHz, Methanol-d4) δ7.63(d,J=8.5Hz,2H),7.33(d,J=8.6Hz,2H),7.14(s,1H),6.91(s,1H),4.47(s,2H),3.83(t,J=6.0Hz,2H),3.67–3. 56(m,16H),3.51(t,J=5.5Hz,2H),3.19(s,4H),2.65(s,3H),2.63(d,J=6 .0Hz,2H),1.94(d,J=8.1Hz,5H),1.79–1.61(m,12H).MS(ESI):m / z[M+H] + 712.4.

[0102] Referring to the above preparation method, the following compounds were prepared:

[0103]

[0104]

[0105]

[0106]

[0107] Example 2

[0108] In this example, the CM1-101 to CM1-112 compounds synthesized in Example 1 were subjected to Western blotting to evaluate their PRMT1 protein degradation abilities in the MCF-7 cell line.

[0109] 1. Experimental Methods

[0110] Cell treatment: Human breast cancer cells (MCF-7) were cultured at 3×10 4 The cells were seeded into 12-well plates at a density of 100 cells / well and incubated overnight in a cell culture incubator at 37°C and 5% CO2. Human breast cancer cells were treated with DMEM complete medium containing 5 μM of the compound for 72 hours, and a DMSO group was set as a negative control.

[0111] Extraction of total cell protein: Remove the culture medium from the culture plate, rinse with cold PBS, then add RIPA lysis buffer containing cocktail protease inhibitor (1:100), and fully lyse the cells at 4°C. Centrifuge at 12000rpm / min at 4°C for 10min, aspirate the supernatant and transfer it to a new 1.5mL centrifuge tube. Measure the concentration of the protein solution using the BCA protein concentration assay kit. Add an equal volume of 2×SDS-PAGE loading buffer to the remaining sample, boil at 96°C for 7min, and load the SDS-PAGE gel after cooling to room temperature.

[0112] Western blot: Use 8-16% SDS-PAGE precast gel to separate the samples by electrophoresis, and the electrophoresis conditions are 120V 60min. The transfer process adopts wet transfer, and the transfer conditions are set to 4℃, 260mA, 90min. After the program is completed, the membrane is transferred to the blocking solution (TBST containing 5% fat-free milk, Tris buffered saline containing Tween 20) and blocked at room temperature for 1h. Cut the band of the target band molecular weight, dilute the primary antibody used for the target protein, the antibody dilution ratio used is 1:5000, and the primary antibody dilution solution also uses TBST containing 5% BSA, and then incubate at 4℃ overnight. After the primary antibody dilution solution is labeled, collect and freeze, and the protein band is washed 3 times with 1×TBST, each time for 10min. Add the corresponding horseradish peroxidase (HRP) labeled secondary antibody, dilute it at a ratio of 1:5000, and incubate it at room temperature for 1h. After labeling the secondary antibody dilution, collect and freeze, wash the protein bands with TBST (Tris buffered saline containing Tween 20) for 3 times, 10 min each time, add ECL chemiluminescent substrate (prepared before use), expose and record.

[0113] 2. Experimental results

[0114] The experimental results are as follows Figure 1 As shown, the compounds, especially CM1-112, can effectively degrade PRMT1 protein in human breast cancer cells.

[0115] Example 3

[0116] Compound CM1-112 was selected to further investigate its ability to degrade PRMT1 protein in human breast cancer cells (MCF-7, MDA-MB-231).

[0117] 1. Experimental Methods

[0118] Cell treatment: Human breast cancer cells (MCF-7, MDA-MB-231) were cultured at 3×10 4 The cells were seeded into 12-well plates at a density of 100 cells / well and incubated overnight in a cell culture incubator at 37°C and 5% CO2. Human breast cancer cells were treated with DMEM complete medium containing compounds of different concentrations for 72 hours, and a DMSO group was set as a negative control.

[0119] The method of Example 1 was used to detect by Western blotting.

[0120] 2. Experimental results

[0121] The experimental results are as follows Figure 2 , Figure 3 As shown, Figure 2 The expression levels of PRMT1 protein in MCF-7 cells after treatment with different concentrations of compound CM1-112 for 72 hours. Figure 3 The expression levels of PRMT1 protein in MDA-MB-231 cells after treatment with different concentrations of compound CM1-112 for 72 hours. The results show that the compounds of the present invention can effectively degrade PRMT1 protein in human breast cancer cells.

[0122] like Figure 4 As shown, the DCs measured by compound CM1-112 in MCF-7 cells 50 =0.83 μM, indicating that compound CM1-112 can effectively inhibit the proliferation of MCF-7 cells.

[0123] Example 4

[0124] Compound CM1-112 was selected to further investigate its ability to inhibit asymmetric dimethylation mediated by endogenous PRMT1 protein in human breast cancer cells (MCF-7, MDA-MB-231).

[0125] 1. Experimental Methods

[0126] Cell treatment: Human breast cancer cells (MCF-7, MDA-MB-231, etc.) were cultured at 5×10 4The cells were inoculated into 12-well plates at a density of 100 / well and incubated overnight in a cell culture incubator at 37°C and 5% CO2. Human breast cancer cells were treated with DMEM complete medium containing compounds with concentration gradients for 48 hours, and a DMSO group was set as a negative control. Western blot experiments were used to detect the inhibitory function of the compounds on the asymmetric dimethylation of Histone H4 protein, a direct substrate of PRMT1, and the asymmetric dimethylation of intracellular proteins.

[0127] 2. Experimental results

[0128] The experimental results are as follows Figure 5 As shown, Figure 5 The results show that compound CM1-112 can effectively inhibit the catalytic function of asymmetric dimethylation of PRMT1 substrate in a concentration gradient-dependent manner.

[0129] Example 5

[0130] Compound CM1-112 was selected to investigate its inhibitory effect on the clonal colonies of human breast cancer cells (MDA-MB-231).

[0131] 1. Experimental Methods

[0132] Cell treatment: Human breast cancer cells (MDA-MB-231) were seeded into 6-well plates at a density of 800 cells / well and incubated overnight in a 37°C, 5% CO2 cell culture incubator. The cells were treated with DMEM complete medium containing 10 μM of the compound for 10 days, 8 days, 6 days, 4 days, and 2 days, and a DMSO group was set as a negative control. The cell clone colonies were stained with crystal violet reagent and the whole body was photographed and recorded.

[0133] 2. Experimental results

[0134] The experimental results are as follows Figure 6 As shown, the number of cell clone colonies formed after treating human breast cancer cells (MDA-MB-231 cell line) with 10 μM compound CM1-112 for different days is shown. The experimental results show that 10 μM compound CM1-112 can effectively inhibit the clone colony formation of human breast cancer cells, effectively inhibit the proliferation of human breast cancer cells and show time-dependent characteristics.

[0135] Example 6

[0136] Compound CM1-112 was selected to investigate its ability to inhibit the proliferation of human breast cancer cells (MDA-MB-231)

[0137] Cell treatment: Human breast cancer cells (MDA-MB-231) were cultured at 5×10 3 The cells were inoculated into 6-well plates at a density of 100 μM / well. After attachment, the cells were treated with DMEM complete medium containing 10 μM compound for 192 hours, and a DMSO group was set as a negative control. The cells were placed in an Incucyte live cell imager (the imager was placed in a 37°C, 5% CO2 cell culture incubator) for incubation. The medium was changed every 48 hours, and the live cell imager was set to take timed photos and record the four fixed field of view areas of the 6-well plate every 12 hours.

[0138] The experimental results are as follows Figure 7 As shown, the changes in cell confluence within 192 hours after treatment of human breast cancer cells (MDA-MB-231 cell line) with 10 μM compound CM1-112 are shown. The confluence data are analyzed using the Incucyte live cell imaging system basic analysis program. The experimental results show that 10 μM compound CM1-112 can effectively slow down the proliferation rate of human breast cancer cells and show time-dependent characteristics.

[0139] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, Linker is selected from: Where Z1 is connected to the benzene ring, Z1 and Z2 are each independently NHC(O) or C(O)NH; n is a positive integer from 1 to 7.

2. The compound according to claim 1, characterized in that Z1 is -NH-CO-, wherein NH is connected to the benzene ring.

3. The compound according to claim 1, characterized in that Z2 is -NH-CO-, wherein CO is connected to the -CH2-adamantane ring.

4. The compound according to claim 1, characterized in that n is 1, 2, 3, 4, 5 or 6.

5. The compound according to claim 1, characterized in that Linker is selected from:

6. The compound according to claim 1, characterized in that The compound is selected from the group consisting of:

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: The compound according to claim 1 or a pharmaceutically acceptable salt thereof; and A pharmaceutically acceptable carrier.

8. Use of the compound or pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 7, characterized in that: Used for preparing PRMT1 degradation agent; or used for preparing medicine for treating diseases related to abnormal expression of PRMT1 protein, preferably used for preparing medicine for treating diseases related to excessive expression of PRMT1 protein.

9. The use according to claim 1, characterized in that The disease is cancer. Preferably, the cancer is selected from the group consisting of breast cancer, gastric cancer, lung cancer, melanoma, and acute myeloid leukemia; more preferably, the cancer is breast cancer.

10. An intermediate of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, having the structure:

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